A device for increasing the depth discrimination is described, e.g., in the German patent application (unexamined first publication) DE 102 50 568 A1 and in the European patent application EP 1 307 774 B1. The present application expressly refers to these two documents. The arrangements and methods described in them are specially suited for use in microscopy, above all in the field of fluorescence microscopy.
For varying the lateral position of the image of the grating-like pattern in the specimen's focal plane, DE 102 50 568 A1 provides for a plane-parallel glass plate, which can be tilted by defined amounts so that the image of the line grating pattern on the specimen shifts within the specimen's focal plane. This can be done, e.g., by means of a galvanic scanner driven via an electronic control system, which controls both the device for increasing the depth discrimination and the optical imaging system. For moving the pattern axially, EP 1 307 774 B1 suggests a motor-driven eccentric motion.
Such devices are implemented, e.g., in the “ApoTome”, a product of Carl Zeiss Microscopy GmbH offered as an optional slide-in module for a number of the company's microscopes.
The use of various objectives—which is readily possible, as a rule, in microscopes with revolving nosepieces, which can accommodate three different objectives, for example—requires that a user has to be furnished with a variety of line grating patterns. A set of grating-like patterns provided will contain one pattern that matches the magnification and numerical aperture of the objective and is best suited to the specific application of generating optical sections. Ideally, this pattern supplies a slice thickness that approximately corresponds to the depth of focus.
In case of a change of objectives, the electronic control system, as a rule, recommends the user which grating-like pattern matches the substituted objective best, this recommendation being the result of computation based on the objective data (in particular, magnification and numerical aperture), or of consulting a look-up table. Selection of the grating-like pattern is made on the basis of the magnification and the numerical aperture of the objective, also taking into account the wavelength of the illuminating light where appropriate. The specifications of the grating-like pattern selected from the set of grating-like patterns by the electronic control system are then displayed to the user on an output unit, e.g., in a dialog box on a monitor. The user should then change the grating-like pattern, but he may also ignore the recommendation.
For changing the grating-like pattern, the user needs to remove the module from the beam path and, subsequently, the line grating from the device before inserting and fixing the other line grating into a suitable holder. As the substrates with the grating-like patterns are rather small, it is necessary, as a rule, to use auxiliary devices for these operations, such as tweezers or similar gripping tools. Thereafter, the module can be reinserted into the microscope.
This time-consuming effort may be just acceptable if objectives have to be changed only occasionally. Where microscopes are provided with objective-changing devices such as, e.g., triple or quintuple revolving nosepieces, which virtually invite a user to frequently alternate between different magnifications, the time and effort involved in changing the grating for structured illumination is disproportionate and all but offsets the advantages offered by the objective changer.